Churchich J E, Wu C
J Biol Chem. 1981 Jan 25;256(2):780-4.
The inhibition kinetic patterns obtained when ATP and pyridoxal analogues are used as inhibitors of the reaction catalyzed by pyridoxal kinase are consistent with a rapid equilibrium random Bi Bi, in which binary complexes, i.e. enzyme . ATP and enzyme . pyridoxal, are formed in kinetically significant amounts. Protein fluorescence quenching was used to determine the dissociation constant (Kd = 25 microM) of ATP . Zn bound to the nucleotide site of the kinase. The binding of ATP to the kinase induces a conformational change which is transmitted to other areas of the macromolecule. Pyridoxaloxime, a competitive inhibitor of pyridoxal, was used as a probe of the pyridoxal-binding site. It binds to the kinase with Ki = 2 microM and displays a fluorescent decay time of 7.8 ns. Time emission anisotropy measurements yield a rotational correlation time for bound pyridoxaloxime of approximately 2 ns, which is considerably shorter than the rotational correlation time of the protein (phi = 38 ns). The fast rotation of pyridoxaloxime remains unaffected by the binding of ATP.
当使用ATP和吡哆醛类似物作为吡哆醛激酶催化反应的抑制剂时，所获得的抑制动力学模式与快速平衡随机双双反应一致，其中二元复合物，即酶·ATP和酶·吡哆醛，以动力学上显著的量形成。利用蛋白质荧光猝灭来确定与激酶核苷酸位点结合的ATP·Zn的解离常数（Kd = 25 μM）。ATP与激酶的结合诱导了构象变化，该变化传递到了大分子的其他区域。吡哆醛肟是吡哆醛的竞争性抑制剂，用作吡哆醛结合位点的探针。它以Ki = 2 μM与激酶结合，荧光衰减时间为7.8 ns。时间发射各向异性测量得出结合的吡哆醛肟的旋转相关时间约为2 ns，这比蛋白质的旋转相关时间（φ = 38 ns）短得多。吡哆醛肟的快速旋转不受ATP结合的影响。
